A metagenomic library cloning strategy that promotes high-level expression of captured genes to enable efficient functional screening.

Functional screening of environmental DNA (eDNA) libraries is a potentially powerful approach to discover enzymatic "unknown unknowns", but is usually heavily biased toward the tiny subset of genes preferentially transcribed and translated by the screening strain. We have overcome this by preparing an eDNA library via partial digest with restriction enzyme FatI (cuts CATG), causing a substantial proportion of ATG start codons to be precisely aligned with strong plasmid-encoded promoter and ribosome-binding sequences. Whereas we were unable to select nitroreductases from standard metagenome libraries, our FatI strategy yielded 21 nitroreductases spanning eight different enzyme families, each conferring resistance to the nitro-antibiotic niclosamide and sensitivity to the nitro-prodrug metronidazole. We showed expression could be improved by co-expressing rare tRNAs and encoded proteins purified directly using an embedded His6-tag. In a transgenic zebrafish model of metronidazole-mediated targeted cell ablation, our lead MhqN-family nitroreductase proved ∼5-fold more effective than the canonical nitroreductase NfsB.

Directed Evolution of the BpsA Carrier Protein Domain for Recognition by Non-cognate 4'-Phosphopantetheinyl Transferases to Enable Inhibitor Screening.

4'-Phosphopantetheinyl transferases (PPTases) play an essential role in activating the carrier protein domains of mega-synthases involved in primary and secondary metabolism and have been validated as promising drug targets in multiple pathogens. Monitoring phosphopantetheinylation of the non-ribosomal peptidase synthetase BpsA, which produces blue indigoidine pigment upon activation, is a useful strategy to screen chemical collections for inhibitors of a target PPTase. However, PPTases can exhibit carrier protein specificity and some medically important PPTases do not activate BpsA. Here, we describe how to conduct a directed evolution campaign to evolve the BpsA carrier protein domain for improved recognition by a candidate PPTase, as exemplified for the human Sfp-like PPTase. This method can be applied to other non-cognate PPTases for discovery of new drug candidates or chemical probes, or to enable development of next-generation biosensors that utilize BpsA as a reporter.

A metagenomic library cloning strategy that promotes high-level expression of captured genes to enable efficient functional screening.

Functional screening of environmental DNA (eDNA) libraries is a potentially powerful approach to discover enzymatic "unknown unknowns", but is usually heavily biased toward the tiny subset of genes preferentially transcribed and translated by the screening strain. We have overcome this by preparing an eDNA library via partial digest with restriction enzyme FatI (cuts CATG), causing a substantial proportion of ATG start codons to be precisely aligned with strong plasmid-encoded promoter and ribosome-binding sequences. Whereas we were unable to select nitroreductases from standard metagenome libraries, our FatI strategy yielded 21 nitroreductases spanning eight different enzyme families, each conferring resistance to the nitro-antibiotic niclosamide and sensitivity to the nitro-prodrug metronidazole. We showed expression could be improved by co-expressing rare tRNAs and encoded proteins purified directly using an embedded His6-tag. In a transgenic zebrafish model of metronidazole-mediated targeted cell ablation, our lead MhqN-family nitroreductase proved ~5-fold more effective than the canonical nitroreductase NfsB.

Inhibition of Indigoidine Synthesis as a High-Throughput Colourimetric Screen for Antibiotics Targeting the Essential Mycobacterium tuberculosis Phosphopantetheinyl Transferase PptT.

A recently-validated and underexplored drug target in Mycobacterium tuberculosis is PptT, an essential phosphopantetheinyl transferase (PPTase) that plays a critical role in activating enzymes for both primary and secondary metabolism. PptT possesses a deep binding pocket that does not readily accept labelled coenzyme A analogues that have previously been used to screen for PPTase inhibitors. Here we report on the development of a high throughput, colourimetric screen that monitors the PptT-mediated activation of the non-ribosomal peptide synthetase BpsA to a blue pigment (indigoidine) synthesising form in vitro. This screen uses unadulterated coenzyme A, avoiding analogues that may interfere with inhibitor binding, and requires only a single-endpoint measurement. We benchmark the screen using the well-characterised Library of Pharmaceutically Active Compounds (LOPAC1280) collection and show that it is both sensitive and able to distinguish weak from strong inhibitors. We further show that the BpsA assay can be applied to quantify the level of inhibition and generate consistent EC50 data. We anticipate these tools will facilitate both the screening of established chemical collections to identify new anti-mycobacterial drug leads and to guide the exploration of structure-activity landscapes to improve existing PPTase inhibitors.

High-Throughput Screening for Inhibitors of the SARS-CoV-2 Protease Using a FRET-Biosensor.

The global SARS-CoV-2 pandemic started late 2019 and currently continues unabated. The lag-time for developing vaccines means it is of paramount importance to be able to quickly develop and repurpose therapeutic drugs. Protein-based biosensors allow screening to be performed using routine molecular laboratory equipment without a need for expensive chemical reagents. Here we present a biosensor for the 3-chymotrypsin-like cysteine protease from SARS-CoV-2, comprising a FRET-capable pair of fluorescent proteins held in proximity by a protease cleavable linker. We demonstrate the utility of this biosensor for inhibitor discovery by screening 1280 compounds from the Library of Pharmaceutically Active Compounds collection. The screening identified 65 inhibitors, with the 20 most active exhibiting sub-micromolar inhibition of 3CLpro in follow-up EC50 assays. The top hits included several compounds not previously identified as 3CLpro inhibitors, in particular five members of a family of aporphine alkaloids that offer promise as new antiviral drug leads.

Directed Evolution of the Nonribosomal Peptide Synthetase BpsA to Enable Recognition by the Human Phosphopantetheinyl Transferase for Counter-Screening Antibiotic Candidates.

Bacterial type II phosphopantetheinyl transferases (PPTases), required for the activation of many cellular mega-synthases, have been validated as promising drug targets in several pathogens. Activation of the blue-pigment-synthesizing nonribosomal peptide synthetase BpsA by a target PPTase can be used to screen in vitro for new antibiotic candidates from chemical libraries. For a complete screening platform, there is a need to also counter-screen inhibitors for cross-reactivity with the endogenous human Type II PPTase (hPPTase), as this is a likely source of toxicity. As hPPTase is unable to recognize the PCP-domain of native BpsA, we used a combination of directed evolution and rational engineering to generate a triple-substitution variant that is able to be efficiently activated by hPPTase. Our engineered BpsA variant was able to readily detect inhibition of both hPPTase and the equivalent rat PPTase by broad-spectrum PPTase inhibitors, demonstrating its potential for high-throughput counter-screening of novel antibiotic candidates.

Mechanistic Understanding Enables the Rational Design of Salicylanilide Combination Therapies for Gram-Negative Infections.

One avenue to combat multidrug-resistant Gram-negative bacteria is the coadministration of multiple drugs (combination therapy), which can be particularly promising if drugs synergize. The identification of synergistic drug combinations, however, is challenging. Detailed understanding of antibiotic mechanisms can address this issue by facilitating the rational design of improved combination therapies. Here, using diverse biochemical and genetic assays, we examine the molecular mechanisms of niclosamide, a clinically approved salicylanilide compound, and demonstrate its potential for Gram-negative combination therapies. We discovered that Gram-negative bacteria possess two innate resistance mechanisms that reduce their niclosamide susceptibility: a primary mechanism mediated by multidrug efflux pumps and a secondary mechanism of nitroreduction. When efflux was compromised, niclosamide became a potent antibiotic, dissipating the proton motive force (PMF), increasing oxidative stress, and reducing ATP production to cause cell death. These insights guided the identification of diverse compounds that synergized with salicylanilides when coadministered (efflux inhibitors, membrane permeabilizers, and antibiotics that are expelled by PMF-dependent efflux), thus suggesting that salicylanilide compounds may have broad utility in combination therapies. We validate these findings in vivo using a murine abscess model, where we show that niclosamide synergizes with the membrane permeabilizing antibiotic colistin against high-density infections of multidrug-resistant Gram-negative clinical isolates. We further demonstrate that enhanced nitroreductase activity is a potential route to adaptive niclosamide resistance but show that this causes collateral susceptibility to clinical nitro-prodrug antibiotics. Thus, we highlight how mechanistic understanding of mode of action, innate/adaptive resistance, and synergy can rationally guide the discovery, development, and stewardship of novel combination therapies.IMPORTANCE There is a critical need for more-effective treatments to combat multidrug-resistant Gram-negative infections. Combination therapies are a promising strategy, especially when these enable existing clinical drugs to be repurposed as antibiotics. We examined the mechanisms of action and basis of innate Gram-negative resistance for the anthelmintic drug niclosamide and subsequently exploited this information to demonstrate that niclosamide and analogs kill Gram-negative bacteria when combined with antibiotics that inhibit drug efflux or permeabilize membranes. We confirm the synergistic potential of niclosamide in vitro against a diverse range of recalcitrant Gram-negative clinical isolates and in vivo in a mouse abscess model. We also demonstrate that nitroreductases can confer resistance to niclosamide but show that evolution of these enzymes for enhanced niclosamide resistance confers a collateral sensitivity to other clinical antibiotics. Our results highlight how detailed mechanistic understanding can accelerate the evaluation and implementation of new combination therapies.

The indigoidine synthetase BpsA provides a colorimetric ATP assay that can be adapted to quantify the substrate preferences of other NRPS enzymes.

OBJECTIVES: To develop a colorimetric assay for ATP based on the blue-pigment synthesising non-ribosomal peptide synthetase (NRPS) BpsA, and to demonstrate its utility in defining the substrate specificity of other NRPS enzymes. RESULTS: BpsA is able to convert two molecules of L-glutamine into the readily-detected blue pigment indigoidine, consuming two molecules of ATP in the process. We showed that the stoichiometry of this reaction is robust and that it can be performed in a microplate format to accurately quantify ATP concentrations to low micromolar levels in a variety of media, using a spectrophotometric plate-reader. We also demonstrated that the assay can be adapted to evaluate the amino acid substrate preferences of NRPS adenylation domains, by adding pyrophosphatase enzyme to drive consumption of ATP in the presence of the preferred substrate. CONCLUSIONS: The robust nature and simplicity of the reaction protocol offers advantages over existing methods for ATP quantification and NRPS substrate analysis.

Structural, functional and evolutionary perspectives on effective re-engineering of non-ribosomal peptide synthetase assembly lines.

Covering: up to May 2018 Non-ribosomal peptide synthetases (NRPSs) are mega-enzymes that form modular templates to assemble specific peptide products, independent of the ribosome. The autonomous nature of the modules in the template offers prospects for re-engineering NRPS enzymes to generate modified peptide products. Although this has clearly been a primary mechanism of natural product diversification throughout evolution, equivalent strategies have proven challenging to implement in the laboratory. In this review we examine key examples of successful and less-successful re-engineering of NRPS templates to generate novel peptides, with the aim of extracting practical guidelines to inform future efforts. We emphasise the importance of maintaining effective protein-protein interactions in recombinant NRPS templates, and identify strengths and limitations of diverse strategies for achieving different engineering outcomes.

Bioactivity-Guided Metabolite Profiling of Feijoa ( Acca sellowiana) Cultivars Identifies 4-Cyclopentene-1,3-dione as a Potent Antifungal Inhibitor of Chitin Synthesis.

Pathogenic fungi continue to develop resistance against current antifungal drugs. To explore the potential of agricultural waste products as a source of novel antifungal compounds, we obtained an unbiased GC-MS profile of 151 compounds from 16 commercial and experimental cultivars of feijoa peels. Multivariate analysis correlated 93% of the compound profiles with antifungal bioactivities. Of the 18 compounds that significantly correlated with antifungal activity, 5 had not previously been described from feijoa. Two novel cultivars were the most bioactive, and the compound 4-cyclopentene-1,3-dione, detected in these cultivars, was potently antifungal (IC50 = 1-2 µM) against human-pathogenic Candida species. Haploinsufficiency and fluorescence microscopy analyses determined that the synthesis of chitin, a fungal-cell-wall polysaccharide, was the target of 4-cyclopentene-1,3-dione. This fungal-specific mechanism was consistent with a 22-70-fold reduction in antibacterial activity. Overall, we identified the agricultural waste product of specific cultivars of feijoa peels as a source of potential high-value antifungal compounds.

A sensitive single-enzyme assay system using the non-ribosomal peptide synthetase BpsA for measurement of L-glutamine in biological samples.

The ability to rapidly, economically and accurately measure L-glutamine concentrations in biological samples is important for many areas of research, medicine or industry, however there is room for improvement on existing methods. We describe here how the enzyme BpsA, a single-module non-ribosomal peptide synthetase able to convert L-glutamine into the blue pigment indigoidine, can be used to accurately measure L-glutamine in biological samples. Although indigoidine has low solubility in aqueous solutions, meaning direct measurements of indigoidine synthesis do not reliably yield linear standard curves, we demonstrate that resolubilisation of the reaction end-products in DMSO overcomes this issue and that spontaneous reduction to colourless leuco-indigoidine occurs too slowly to interfere with assay accuracy. Our protocol is amenable to a 96-well microtitre format and can be used to measure L-glutamine in common bacterial and mammalian culture media, urine, and deproteinated plasma. We show that active BpsA can be prepared in high yield by expressing it in the apo-form to avoid the toxicity of indigoidine to Escherichia coli host cells, then activating it to the holo-form in cell lysates prior to purification; and that BpsA has a lengthy shelf-life, retaining >95% activity when stored at either -20 °C or 4 °C for 24 weeks.

Crystal structure of the essential Mycobacterium tuberculosis phosphopantetheinyl transferase PptT, solved as a fusion protein with maltose binding protein.

Phosphopantetheinyl transferases (PPTases) are key enzymes in the assembly-line production of complex molecules such as fatty acids, polyketides and polypeptides, where they activate acyl or peptidyl carrier proteins, transferring a 4'-phosphopantetheinyl moiety from coenzyme A (CoA) to a reactive serine residue on the carrier protein. The human pathogen Mycobacterium tuberculosis encodes two PPTases, both essential and therefore attractive drug targets. We report the structure of the type-II PPTase PptT, obtained from crystals of a fusion protein with maltose binding protein. The structure, at 1.75Å resolution (R=0.156, Rfree=0.191), reveals an α/ß fold broadly similar to other type-II PPTases, but with differences in peripheral structural elements. A bound CoA is clearly defined with its pantetheinyl arm tucked into a hydrophobic pocket. Interactions involving the CoA diphosphate, bound Mg(2+) and three active site acidic side chains suggest a plausible pathway for proton transfer during catalysis.